Molecular dissection of the chicken Toll-like receptor repertoire

Abstract

Toll-like receptors (TLRs) are host immune sensors that continuously search for the presence of microbial pathogens. TLRs play a key role in the innate defense against pathogens and the development of adaptive immunity, and are important determinants of the susceptibility to infections. The receptors are present in virtually all species, including insects, amphibians, fish, birds and mammals. Yet, the activity of the TLR family in the different species is variable and unpredictable. The TLRs are divided in different families based on homology, ligand specificity, function and cellular localization. TLR2 together with either TLR1 or TLR6 recognizes tri- and di-acylated lipopeptides, respectively. Bacterial lipopolysaccharide (LPS) is recognized by the TLR4/MD-2 complex, and TLR5 is the receptor for bacterial flagellin. TLR3, TLR7, TLR8 and TLR9 are expressed intracellularly and are activated by dsRNA (TLR3), ssRNA (TLR7 and TLR8) and bacterial DNA (TLR9). Activation of TLRs results in the production of antimicrobial peptides and pro-inflammatory and immunomodulating molecules (cytokines, chemokines) that give rise to an effective antimicrobial response and direct the adaptive immune system. The present study was designed to determine the function of the chicken TLR repertoire. Chicken often carry infectious agents that cause human disease. Knowledge of the chicken innate defense against these pathogens may provide important clues as to how to eradicate these pathogens from chicken. Genome analysis indicates that chicken may express orthologs of TLR1/6/10, TLR2 (type 1 and type 2), TLR3, TLR4, TLR5 and TLR7. Two types of TLRs, TLR15 and TLR21, appear to absent in mammals. We cloned the chicken TLRs and tested their functions using the major foodborne pathogen Salmonella as a model infectious agent. In Chapter 2, we show that expression of chTLR2 type 2 together with the TLR1/6/10 ortholog, which we termed chTLR16, induces a potent innate immune response towards both di-and tri-acylated lipopeptides. The chTLR2/chTLR16 complex thus combines the functions of the mammalian TLR2/6 and TLR2/1 heterodimers in a single TLR complex. For chTLR4 we discovered that this TLR in conjunction with chMD-2 responds to LPS. The complex formation between chTLR4 and chMD-2 is species-specific (Chapter 3). Of particular interest, the chTLR4 complex failed to activate one of the signaling pathways that is activated by the mammalian TLR4 complex, which may explain the relative resistance of chicken to LPS. The chTLR5 receptor responds to Salmonella flagellin (Chapter 4). Targeted mutagenesis of the flagellin protein showed species-specific recognition of the flagellin compared to the human and mouse TLR5. Chapter 5 reports the elucidation of the function of chTLR21 which is absent in mammalian species. This receptor responds to CpG DNA but with broader specificity than human or murine TLR9. Our results provide evidence that the chicken TLR system is functional and has several unique properties both at the level of ligand specificity, the formation of TLR receptor complexes, and activated TLR signaling pathways. This knowledge opens novel opportunities for rational design of the much needed vaccines against major bacterial foodborne pathogens, thus contributing to food safety.